1. Field of the Invention
This invention relates to polymerized liposomes which are linked to a targeting agent and may also be linked to at least one of an image contrast enhancement agent and a therapeutic or treatment agent to provide targeted polymerized liposome diagnostic agents and targeted polymerized liposome therapeutic agents, respectively. In one embodiment, this invention relates to liposomes which may be linked to contrast ions for magnetic resonance imaging and radioisotope imaging or optical imaging by using chromophores attached to the liposome or chromophores inherent in the particle in which the polymerization adds stability in vivo. The paramagnetic or radioactive polymerized liposomes may also be linked to antibodies and ligands for specific interaction with biological targets holding the contrast agent to specific biological sites, providing in vitro and in vivo study of the expression of molecules in or on the surface of cells and tissues during disease and pathology. In another embodiment, targeted polymerized liposomes may be linked to or encapsulate a therapeutic agent, such as, for example, proteins, hormones and drugs, for directed delivery of a treatment agent to specific biological locations for localized treatment.
2. Description of Related Art
Liposomes have been used as carriers for administration of drugs and paramagnetic contrast agents. U.S. Pat. Nos. 5,077,057 and 5,277,914 teach preparation of liposome or lipidic particle suspensions having particles of a defined size, particularly lipids soluble in an aprotic solvent, for delivery of drugs having poor aqueous solubility. U.S. Pat. No. 4,544,545 teaches phospholipid liposomes having an outer layer including a modified cholesterol derivative to render the liposome more specific for a preselected organ. U.S. Pat. No. 5,213,804 teaches liposome compositions containing an entrapped agent, such as a drug, which are composed of vesicle-forming lipids and 1 to 20 mole percent of a vesicle-forming lipid derivitized with hydrophilic biocompatible polymer and sized to control its biodistribution and recirculatory half life. U.S. Pat. No. 5,246,707 teaches phospholipid coated microcrystalline particles of bio-active material to control the rate of release of entrapped water soluble biomolecules, such as proteins and polypeptides. U.S. Pat. No. 5,158,760 teaches liposome encapsulated radio-active labeled proteins, such as hemoglobin.
The use of magnetic resonance imaging contrast enhancement agents or radioactive isotopes in the body is practiced by a variety of methods. U.S. Pat. No. 5,135,737 teaches magnetic resonance imaging enhancement agents of paramagnetic metal ion chelates attached to polymers such as polyamine based molecules with antibodies attached for concentration at desired sites in the body. U.S. Pat. Nos. 4,938,947 and 5,017,359 teach an aerosol composition containing soluble fragments of bacterial wall or cell peptidoglycan which may be labeled with a paramagnetic element and encapsulated in liposomes which may be administered as an aerosol. U.S. Pat. No. 5,078,986 teaches magnetic resonance imaging agents of a chelate of a paramagnetic element carried by or within the external surface of a liposome and released at a desired organ or tissue site. PCT Publication Number WO 92/21017 teaches specific liposomes complexed with paramagnetic ions to prolong their blood pool half life and control magnetic resonance relaxivity. Liposomes as MR contrast agents has been reviewed by Unger, E. C., Shen, D. K., and Fritz, T. A., Status of Liposomes as MR Contrast Agents, JMRI, 3, 195-198, (1993).
The need for recirculation of paramagnetic contrast agents in the body, that is avoidance of rapid endocytosis by the. reticuloendothelial system and avoidance of rapid filtration by the kidney, to provide sufficient concentration at a targeted site to afford necessary contrast has been recognized. The use of small molecules, such as gadolinium diethylenetriaminepentaacetic acid, is restricted due to rapid renal excretion while most liposomes, having diameters  greater than 800 nm, are quickly cleared by the reticuloendothelial system. Attempts to solve these problems have involved use of macromolecular materials, such as gadolinium diethylenetriaminepentaacetic acid derived polysaccharides, polypeptides, and proteins. These agents have not achieved the versatility in chemical modification to provide for both long recirculation times and active targeting.
Prior attempts to construct bifunctional, ligand-bearing magnetic resonance contrast agents have not been satisfactory due to insufficient sensitivity, poor target specificity and lack of characterization. Gore, J. C. and Smith, F. W., Special Issue: Contrast Agents, Magn. Reson. Img., 3, 1-97, (1985); Hasso, A. N. and Stark, D. D., Special Issue: Contrast Agents, JMRI, 3, 137-310, (1993); and Wehrli, F. W., SMRM Workshop: Contrast Enhanced Magnetic Resonance, Magn. Reson. Med., 22, 177-378, (1991).
Receptor-directed contrast agents for MRI have been attempted using iron oxide particles, but the chemistry and characterization of the particle has been poorly defined and thus it has been difficult to achieve control over non-specific adhesion, blood pool half life and the versatility for both T1 and T2* imaging modes. In addition, no radioisotope imaging is possible using these iron-based agents which further limits their usefulness. Reimer, P., Weissleder, R., Brady, T. J., Baldwin, B. H., Tennant, B. C., and Wittenberg, J., Experimental Hepatocellular Carcinoma: MR Receptor Imaging, Radiology, 180, 641-645 (1991), Reimer, P., Weisslender, R., Lee, A. S., and Brady, T. J., Receptor Imaging: Application to MR Imaging of Liver Cancer, Radiology, 177, 729-734 (1990), Reimer, P., Weissleder, R., Wittenberg, J., and Brady, T. J., Receptor-Directed Contrast Agents for MR Imaging: Preclinical Evaluation With Affinity Assays, Radiology, 182, 565-569 (1992), and Weissleder, R., Reimer, P., Lee, A. S., Wittenberg, J. and Brady, T. J., MR Receptor Imaging: Ultrasmall Iron Oxide Particles Targeted to Asialoglycoprotein Receptors, AJR, 155, 1161-67, (1990).
Antibody MR imaging has been described by Unger, E. C., Totty, W. G., Neufeld, D. M., Otsuka, F. L., Murphy, W. A., Welch, M. S., Connett, J. M., and Philpott, G. W., Magnetic Resonance Imaging Using Gadolinium labeled Monoclonal Antibody, Invest. Radiol., 20, 693-700. (1985), and Weissleder, R., Lee, A. S., Fischman, A. J., Reimer, P., Shen, T., Wilkinson, R., Callahan, R. J., and Brady, T. J., Polyclonal Human Immunoglobulin G Labeled with polymeric Iron Oxide: Antibody MR Imaging, Radiology, 181, 245-249, (1991). In the former case, one is limited by the amount of contrast enhancement that can be achieved by direct attachment of chelator to an antibody. In the latter case, the iron oxide particle is not amenable to control over surface functionality needed to reduce non specific adhesion and the particle is not well characterized or well tolerated in vivo.
The economic driven requirement for improved in vitro diagnostic techniques for medicine is also well recognized. Hannon, Robert E., Future Practices in Diagnostic Medicine, Arch Pathol Lab Med, Vol 119, pg 890-893 (October 1995) A common technique presently used in diagnostic medicine for detection of the presence of specific antigens in solution is addition of latex beads coated with antibodies to the solution and detection of micro-agglutinated products, as described in Microparticle Immunoassay Techniches, 2nd Ed., Seradyn, Inc., Particle Technology Division, P.O. Box 1210, Indianapolis, Ind. 46206 (1993) and U.S. Pat. Nos. 4,801,504 and 5,053,443. However, detection of micro-agglutinated products, approximately 1 xcexcm in size, is very difficult.
Currently used in vitro enzyme linked immunoassays (ELISA) have a sensitivity in the order of 1 picomolar concentration (0.5 xcexcg/10 mL). Other in vitro assay technologies, including radioactive immunoassay systems, have sensitivities 2 to 3 orders of magnitude more sensitive than ELISA assays. While polymerase chain reaction (PCR) based technologies have the technology is limited to detection of nucleic acids.
The expression of glycoproteins on a cell surface is currently detected using assays requiring multiple steps and frequently resulting in low sensitivity. For example, for assays of protein expression on activated endothelial cells, a first step involves the use of an antibody against the cell surface protein followed by multiple steps to amplify the ability for detection of the resulting complexes using flourescent techniques, such as, for example, fluorescent antivated cell flow cytometry, fluorescent antivated cell sorting, and fluorescent microscopy.
It has been recognized that unique proteins called cell adhesion molecules (CAMs) are expressed by endothelial cells during a variety of physiological and disease processes. Reisfeld, R. A., Monoclonal Antibodies in Cancer Immunotherapy, Laboratory Immunology II, Vol. 12, No. 2, pgs. 201-216, (June 1992) and Archelos, J. J., Jung, S., Maurer, M., Schmied, M., Lassmann, H., Tamatani, T., Miyasaka, M., Toyka, K. V. and Hartung, H. P., Inhibition of Experimental Autoimmune Encephalomylitis by the Anitbody to the Intercelluler Adhesion Molecule ICAM-1, Ann. of Neurology, Vol. 34, No. 2, pgs. 145-154 (1993) Multiple endothelial ligands and receptors, including CAMs, are known to be upregulated during various pathologies, such as inflammation and neoplasia. Currently, the evaluation of the pathophysiology of the cell adhesion molecules is generally limited to in vitro assays.
This invention, in one embodiment, relates to nanoscale polymerized liposome particles based upon lipids having a polymerizable functional group and a metal chelator to attach an imaging enhancement agent, such as paramagne tic or radioactive ions, which assemble to form imaging enhancement polymerized liposomes. In preferred embodiments, the imaging enhancement polymerized liposomes are derivatized with antibodies and/or ligands for in vivo binding to cell surface receptors of targeted cells. In particular, these receptors can be located on the endothelium which eliminates the need for distribution of the active agent out of the blood pool. Paramagnetic polymerized liposomes according to this invention have been found to be well tolerated by rabbits, mice and rats, even on repeated administration, and effectively recirculate in the bloodstream, avoiding rapid endocytosis by the reticuloendothelial system. These materials provide good magnetic resonance imaging signal enhancement of targeted cells, liver and kidney, for long periods of time, of 90 minutes and more.
The polymerized liposomes of this invention are stable in vivo and provide for effective control of particle size, surface functionality, active ion density and water accessibility to maximize their effective relaxivity for T1 and T2* magnetic resonance imaging enhancement of specific biological systems. For example, the polymerized liposomes of this invention may have a plurality of metal ions for high relaxivity per particle providing highly effective magnetic resonance imaging enhancement and may also have attached antibodies or ligands specific for cellular receptors, resulting in a sensitive probe for areas of vascular tissue expressing these cell surface molecules. Receptors of protein adhesins on the endothelium surface are of particular interest in this targeting scheme because they are expressed extensively during pathological processes of inflammation for the recruitment of leukocytes or in the process of angiogenesis for vascularization of diseased tissue, such as tumors. Targeted polymerized liposomes provide for in vivo magnetic resonance imaging histology that enables early evaluation of changes in the endothelium in disease processes due to the attachment of a high concentration of paramagnetic or superparamagnetic ions to specific receptors on specifically targeted tissue or endothelium of concern.
This invention provides various methods for in vitro assays. For example, antibody-conjugated polymerized liposomes, according to this invention, provide an ultra-sensitive diagnostic assay for specific antigens in solution. Polymerized liposomes of this invention having a chelator head group chelated to spectroscopically distinct ions provide high sensitivity for enzyme linked immunoassays. Polymerized liposomes of this invention having a fluorophore head group provide a method for detection of glycoproteins on cell surfaces.
In one embodiment of this invention, a targeting polymerized liposome particle comprises: an assembly of a plurality of liposome forming lipids each having an active hydrophilic head group linked by a bifunctional linker portion to the liposome forming lipid, and a hydrophobic tail group having a polymerizable functional group polymerized with a polymerizable functional group of an adjacent hydrophobic tail group of one of the plurality of liposome forming lipids, at least a portion of the hydrophilic head groups having an attached targeting active agent for attachment to a specific biological molecule. In another embodiment, the targeting polymerized liposome particle has a second portion of the hydrophilic head groups with functional surface groups attached to an image contrast enhancement agent to form a targeting image enhancing polymerized liposome particle. In yet another embodiment, a portion of the hydrophilic head groups have functional surface groups attached to or encapsulating a treatment agent for interaction with a biological site at or near the specific biological molecule to which the particle attaches, forming a targeting delivery polymerized liposome particle or a targeting image enhancing delivery polymerized liposome particle.
This invention provides a method of assaying abnormal pathology in vitro comprising, introducing a plurality of targeting polymerized liposome particles targeted to a molecule involved in the abnormal pathology into a fluid contacting the abnormal pathology, the targeting polymerized liposome particles attaching to a molecule involved in the abnormal pathology, and detecting in vitro the targeting polymerized liposome particles attached to molecules involved in the abnormal pathology.
This invention also provides a method of diagnosing abnormal pathology in vivo comprising, introducing a plurality of targeting image enhancing polymerized particles targeted to a molecule involved in the abnormal pathology into a bodily fluid contacting the abnormal pathology, the targeting image enhancing polymerized particles attaching to a molecule involved in the abnormal pathology, and imaging in vivo the targeting image enhancing polymerized particles attached to molecules involved in the abnormal pathology.
This invention further provides a method of therapeutic treatment comprising, introducing into a bodily fluid contacting an area of desired treatment a plurality of targeting delivery polymerized liposome particles targeted to a molecule at or near the site of desired treatment and having a desired therapeutic agent attached or encapsulated, the targeting delivery polymerized liposome particles attaching to molecules at or near the site of desired treatment rendering the therapeutic agent available at the site of desired treatment.
Further details of preparation and use of the targeting paramagnetic polymerized liposomes of this invention in magnetic resonance imaging is described in: Storrs, R. W., Tropper, F. D., Li, H. Y., Song, C. K., Kuniyoshi, J. K., Sipkins, D. A., Li, K. C. P. and Bednarski, M. D., Paramagnetic Polymerized Liposomes: Synthesis, Characterization, and Applications for Magnetic Resonance Imaging, J. Am. Chem. Soc., Vol. 117, No. 28, pgs. 7301-7306, (August 1995) and Storrs, R. W., Tropper, F. D., Li, H. Y., Song, C. K., Sipkins, D. A., Kuniyoshi, J. K., Bednarski, M. D., Strauss, H. W. and Li, K. C. P., Paramagnetic Polymerized Liposomes as New Recirculating MR Contrast Agents, JMRI, Vol. 5, No. 6, pgs.719-724, (November/December 1995), which publications are incorporated herein by reference in their entireties.